Targeted Modification of Membrane Lipids Project Summary The lipid composition of membranes has critical ramifications in biology. It has been long known that bacterial and mammalian cells harbor a different set of lipids in their membranes. While a mammalian cell membrane is largely composed of zwitterionic lipids, bacterial cells typically display anionic lipids in large quantities. Taking advantage of this difference, many organisms have evolved cationic host defense peptides (HDPs), which serve as the frontline of the innate immunity to fend off invading bacterial pathogens. For example, human neutrophils rely on cationic defensins for bacterial cell killing and clearance. To acquire resistance against cationic HDPs, selected bacterial species synthesize the lipid Lys-PG, which carries a net positive charge. We hypothesize that synthetic molecules that bind Lys-PG and consequently mask its net charge will re-sensitize the bacterial cells to killing by HDPs. To test the hypothesis, we will develop synthetic modifiers of Lys-PG by introducing reversible covalent warheads into well-structured scaffolds. Further we will test the efficacy of Lys-PG modification in vitro and in mouse infection models. The specific aims are: 1) we will use a known, foldable cyclic peptide scaffold to assemble multiple side chains for Lys-PG modification. Computational modeling will be integrated with experimental characterization to optimize for Lys-PG binding; 2) we will develop potent and specific modifiers of Lys-PG by screening novel bicyclic peptide libraries. This part of the proposal will be based on a powerful peptide bicyclization strategy recently developed by our group; 3) the Lys-PG modifiers developed in 1) and 2) will be tested for their capability to potentiate the bactericidal activity of several HDPs and neutrophils. Their efficacy to facilitate bacterial clearance will be further examined in mouse models of infection. With success, the proposed work will yield a novel strategy to fight against the drug-resistance strains of bacterial pathogens. Although the proposed work focuses on Lys-PG, the methodology developed here should be extendable to other lipid modifications of biological significance.